Stem cells replenish the cells lost in tissues due to turnover or trauma. In addition there is increasing evidence that several diseases, including some associated with aging and also many forms of cancer, are caused by inappropriate regulation of stem cells. Thus on one hand, understanding stem cell biology has very broad implications for manipulating stem cells for therapeutic purposes and, on the other hand, giving modern medicine new targets to combat a host of very high impact diseases. The studies supported by this award have produced a detailed model for the maintenance and differentiation of the germline stem cell during oogenesis in Drosophila. The germline stem cell niche has proven itself to be a valuable model for understanding the signaling and gene regulation that selects stem cells and guides their differentiation into functional gametes. The bag-of-marbles (bam) gene has been a primary focus of these studies because of the primary role it plays in controlling the stem cell-to-cystoblast transition. Niche signaling specifically silences bam transcription and the cystoblast expression program is blocked. When the stem cell produces a daughter cell that moves out of the signaling niche, bam expression is derepressed and cystoblast differentiation becomes activated. The most recent studies defined additional factors that control bam silencing but, even more importantly, have revealed several genes that are required for germ line stem cell maintenance without acting directly on bam transcription. These have therefore revealed novel aspects of the regulatory pathways regulating stem cell fate. The Pumilio-Nanos translational inhibitory complex is especially important in this regard and the most recent findings show that cystoblast differentiation begins because Bam antagonizes the action of the Pumilio- Nanos complex by blocking translation of Nanos. In the studies proposed in this application we seek to extend these discoveries to a refined molecular mechanism explaining how Bam inhibits Nanos translation. The proposed studies will: (1) define the Bam Response Elements that reside in the Nanos 3'-UTR. These experiments will use classical deletion and transgenic constructs strategies to test the biological activity of experimental transgenes (2) carry out genetic modifier screens to identify the factors that work with Bam and/or that regulate cystoblast transition downstream of bam (3) mount a comprehensive strategy to identify the polypeptides that interact with Bam to form the complex that antagonizes the action of Pumilio-Nanos. The goals of this Aim will use yeast two-hybrid and co- immunoprecipitation methods to identify components of the Bam polypeptide complex. Public Health Relevance: Stem cells offer great therapeutic and research opportunities but their biology is poorly understood. Studies of the Drosophila germ line stem cells and the stromal cells that support them have revealed important features of the signaling and gene expression programs that regulate stem cells. The studies proposed in this application will probe new ideas about the factors and processes that control stem cells and the differentiation of their progeny.